Protein folding is a complex process, and many disease-associated mutations impair the ability of the protein to attain the proper 3-dimensional native conformation. Terminally misfolded proteins must be triaged for degradation to avoid cytotoxic accumulation of misfolded proteins. ER-associated degradation (ERAD) is an essential quality control process that recognizes and degrades terminally misfolded secretory and transmembrane proteins, and is critical for cellular homeostasis. The protein machinery and molecular mechanisms that are involved are only now beginning to emerge. The long-term objective of this proposal is to determine the cis- and trans- factors underlying substrate specific ERAD. The immediate goals of this project are to 1) Characterize the molecular features responsible for substrate specificity of known ERAD components;2) Identify novel UPS components involved in ERAD;3) Characterize the molecular role of the identified UPS components in ERAD. To characterize the molecular features mediating substrate-specific degradation and to define the components involved in substrate-specific ERAD pathways, known ERAD components will be depleted using RNA interference and the effect will be analyzed using a panel of topologically distinct, fluorescently tagged ERAD substrates. In order to systematically identify novel ERAD machinery, a functional genomic screen employing a small hairpin RNA (shRNA) library targeting all known and predicted components of the UPS will be performed using high-throughput flow cytometry. Positive hits will be subjected to extensive validation and characterized using a battery of cellular and biochemical assays to determine their role in ERAD, including assessment of substrate degradation kinetics, ubiquitination, glycosylation, translocation, and aggregation. Together these studies will yield insight into the substrate features mediating specificity in ERAD and the identity of the components involved in ERAD. Deficits in ERAD have been implicated as a underlying cause for many human diseases, including lung disease, liver disease, cancer, diabetes, and several neurodegenerative diseases. The proteins and molecular mechanisms that regulate this pathway are poorly understood and represent an important area of research that will directly impact our understanding of a broad spectrum of diseases. Completion of this project will identify new proteins and insights into the ERAD process, and may yield clinically relevant targets and strategies for the development of mechanism-based therapeutics.